Many areas of basic research benefit from the ability to rapidly and sensitively detect nucleic acids. For example, in many fields of life sciences research, including biological, biomedical, genetic, fermentation, aquaculture, agriculture, forensic and environmental research, there is a need to identify nucleic acids both within and without cells as a routine component of standard experimental methods. A common example is the widespread use of gel electrophoresis for characterizing nucleic acids, one limitation of which is the sensitivity of the staining method used to detect the nucleic acid bands.
In the life and medical sciences, researchers and technicians often need to identify intracellular nucleic acids and/or sort cells based on the quantity of nucleic acids present in the cells. The quantity of nucleic acids present can be indicative of the type of cells, or even the presence of disease states in cells (e.g., nucleated human erythrocytes). Such applications require a fast, sensitive and selective methodology that can detect nucleic acids, even when bounded (or surrounded) by cellular membranes.
Dyes that are generally applicable for staining nucleic acids across a broad range of applications preferably have the following properties:
i) the nucleic acid-dye complex should produce a very high signal with low background so that small quantities of nucleic acids can be sensitively detected in both cell-free and cell-based assays; and PA1 ii) the nucleic acid-dye complex should be photostable so that the fluorescent signal may be observed, monitored and recorded without significant photo bleaching. PA1 iii) the dye should be permeable to cell membranes so that it can bind nucleic acids sequestered in cells; PA1 iv) the membrane permeation kinetics should be relatively fast so that detectable signals can be obtained upon relatively brief exposures to the dye; and PA1 v) the dye should be non-toxic to living cells so that staining will not disrupt the normal metabolic processes of the cells or cause premature cell death.
For applications involving staining nucleic acids in cells, especially live cells, the dyes should preferably have the following additional properties:
A variety of dyes useful for staining nucleic acids in cell-free and/or intracellular assays have been described. For example, a variety of asymmetrical cyanine dyes (Brooker et al., 1942, J. Am. Chem. Soc. 64:199) and thioflavin dyes (U.S. Pat. Nos. 4,554,546 and 5,057,413) useful for staining nucleic acids have been described. The non-chimeric asymmetrical cyanine dye sold under the trade name Thiazole Orange provides particular advantages in the quantitative analysis of immature blood cells or reticulocytes (U.S. Pat. No. 4,883,867) and in preferentially staining nucleic acids of blood-borne parasites (U.S. Pat. No. 4,937,198). Although Thiazole Orange and other thioflavin cyanine dyes are permeable to membranes of many mammalian cells, they are non-permeable to many eukaryotic cells.
Other related cyanine dyes have been described which are non-permeable to living cells unless their membranes have been disrupted (see, U.S. Pat. Nos. 5,321,130 and 5,410,030). A variety of dimeric dyes having cationic moicties useful for staining nucleic acids in electrophoretic gels are described in U.S. Pat. Nos. 5,312,921; 5,401,847; 5,565,554; and 5,783,687.
Substituted asymmetric cyanine dyes capable of permeating membranes of a broad spectrum of both living and dead cells have also been described (see, U.S. Pat. No. 5,436,134).
While many of these dyes have found use as nucleic acid stains, they suffer several drawbacks which limit their general applicability, particularly in live-cell assays. For example, most of the available dyes fluoresce in the green region of the visible spectrum. Not only are green lasers more expensive than red lasers, green fluorescence results in higher background signals in live cell assays due to, among other factors, autofluorescence of cellular components and assay equipment. These higher background signals decrease the sensitivity of the assay. Moreover, many cellular components absorb green light, further reducing the sensitivity of the assay.
Since red lasers are less expensive than green lasers and cellular components are generally transparent to red light, nucleic acid stains that have excitation and emission maxima in the red region of the visible spectrum are preferred for live-cell assays. However, the availability of membrane permeable red-emitting nucleic acid stains having suitable properties for live-cell assays is limited. Unfortunately, the most common water-soluble red-emitting dyes, the cyanine dyes such as dye Cy5, are not photostable. Thus, sensitive nucleic acid stains that are photostable, have excitation and emission maxima in the red region of the visible spectrum and that are permeable to cell membranes are highly desirable.